Urinary tract infection among people living with human immunodeficiency virus attending selected hospitals in Addis Ababa and Adama, central Ethiopia

Background Urinary tract infections (UTIs) and antibacterial resistance (ABR) are important public health problems, but they are not well-studied among people living with human immunodeficiency virus (PLHIV) globally, especially in low-income countries. Therefore, it is important to regularly measure the extent of UTIs and ABR in the most susceptible populations. This study aimed to investigate the prevalence of UTIs, associated factors, bacterial causal agents, and their antibiotic susceptibility profile among PLHIV in central Ethiopia. Methods A hospital-based cross-sectional study was conducted to recruit 688 PLHIV by a simple random sampling method. Background information was gathered through interviews, while clinical information was gathered from recent information sheets of patient charts using organized, pretested, and validated study tools. Midstream urine was collected aseptically and transported to the Microbiology Laboratory of Aklilu Lemma Institute of Pathobiology within 4 h of collection, maintaining its cold chain. Standard conventional microbial culture methods and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry were used to identify the bacterial isolates at the species level. Kirby Bauer’s disk diffusion method was used to determine the antibiotic susceptibility profile of the bacterial isolates based on the interpretation guidelines of the Clinical Laboratory Standard Institute. Logistic regression models were used to examine factors associated with the occurrence of UTIs among PLHIV attending selected hospitals in Addis Ababa, and Adama. Results Out of 688 PLHIVs involved in the current study, 144 (20.9%) were positive for UTIs, whereas the majority were asymptomatic for UTIs. In the multivariable logistic regression analysis, only HIV RNA ≥ 200 copies/ml [AOR = 12.24 (95% CI, 3.24, 46.20), p < 0.01] and being symptomatic for UTIs during the study period [AOR = 11.57 (95% CI, 5.83, 22.97), p < 0.01] were associated with the occurrence of UTIs. The dominant bacterial species isolated were Escherichia coli (E. coli; n = 65; 43%), followed by Enterococcus faecalis (E. faecalis; n = 16; 10.6%) and Klebsiella pneumoniae (K. pneumoniae; n = 11; 7.3%). Over half of the E. coli isolates were resistant to antibiotics such as gentamicin (GM; n = 44; 67.7%), amikacin (AN; n = 46; 70.8%), nalidixic acid (NA; n = 42; 64.6%), ciprofloxacin (CIP; n = 40; 61.5%), and azithromycin (AZM; n = 45; 69.2%). All of the K. pneumoniae isolates (n = 11; 100%), (n = 6; 54.5%), and (n = 7; 63.6%) were resistant to [amoxicillin as well as amoxicillin + clavulanic acid], ceftriaxone, and sulfamethoxazole + trimethoprim, respectively. All the Staphylococcus aureus (S. aureus) isolates were resistant to cefoxitin, which implies methicillin-resistant S. aureus (MRSA). Conclusion The high prevalence of UTIs and antibiotic resistance revealed in the current study needs public health interventions such as educating the population about preventive measures and the importance of early treatment of UTIs. Our findings also highlight the need to provide UTI screening services for PLHIV, and healthcare providers should adopt antibiotic stewardship programs to promote and ensure their appropriate and judicious use.


Background
Urinary tract infections are highly prevalent worldwide, with approximately 150 million cases annually (1) among people of all ages; however, they are higher among youngsters, elders, and females (2).The infection poses significant public health concerns among PLHIV, mainly in developing countries (2,3).This could be due to the suppressed immunity of the population and limited healthcare services in the region, which results in a high economic burden and reduced quality of life (3,4).Untreated UTIs can lead to severe complications (5) such as pyelonephritis, renal failure, prostatitis, epididymitis, septicemia, endocarditis, osteomyelitis, septic arthritis, and meningitis (6,7) which could be severe and sometimes life-threatening (6).
The pathogenesis of the infection involves the bacterial coliforms inhabiting the urethral introitus and ascending into the bladder or kidneys (5), attaching to the urothelium with the help of their microbial adhesins, and binding to specific receptors on the host cells (2,3,5).Moreover, quorum sensing, a form of bacterial communication, plays a significant role in UTI pathogenesis.Bacteria communicate via signaling molecules to regulate gene expression, coordinate virulence factor production, and biofilm formation (2,3).This helps sense population density and initiate actions, such as producing proteases, toxins, urease enzymes, and ironscavenging systems, contributing to infection invasion and establishment of infection (3), which is manifested by flank pain, dysuria, pyuria, increased urinary frequency/urgency, and fever and chills if the infection involves the kidneys or when it is more severe (17).Furthermore, the diagnosis of UTIs is based on urine analysis for white blood cells and urine culture to identify bacteria and antibiotic susceptibility (18).However, antibiotic resistance challenges the effectiveness of antimicrobials, mainly in developing countries, due to inappropriate antibiotic use and poor antimicrobial stewardship programs (3).
UTIs represent a significant concern among PLHIV, with varying prevalence worldwide and in developing countries (19,20).For instance, a systematic review and meta-analysis from Ethiopia revealed a 12.8% pooled prevalence of UTIs among PLHIV (20), which was supported by findings reported from the region, which ranges from 10.3 to 48.7% (4,9,21,22).The burden of UTIs is impacted by the socio-demographic, background, and clinical characteristics of the population (4,20,23).The common causal agents of UTIs revealed by studies from developing countries, including Ethiopia, are E. coli, S. aureus, K. pneumoniae, Enterococcus species, and Enterobacter aerogenes, which is in agreement with global reports (9,21,(24)(25)(26).Bacterial uropathogens among PLHIV usually demonstrated a higher rate of ABR (27), which is intense in low-income countries (27).Studies from low-income countries, including Ethiopia, revealed a higher rate of MDR, ranging from 45.9 to 95% (4,19,(26)(27)(28)(29).Thus, it is important to assess the extent of UTIs, and ABR associated with bacterial uropathogens, along with suggestions of possible interventions.Therefore, this study aimed to determine the prevalence of UTI, its associated factors, and the antibacterial susceptibility profile of the bacterial isolates among PLHIV attending selected antiretroviral therapy (ART) clinics in Addis Ababa and Adama, central Ethiopia.

Study setting, and study design
A hospital-based cross-sectional study was conducted in central Ethiopia (Addis Ababa and Adama cities) among PLHIV from October 2022 to April 2023.Addis Ababa, the nation's capital city, is located at a high altitude, 2,400 meters above sea level (30), while Adama is located 99 km southeast of Addis Ababa (31), in the great East African rift valley system, 08°32′29″N 39°16′08″E; 1712 meters above sea level (31).According to the world population review estimates, the population of Addis Ababa and Adama by the year 2024 would be 3.6 million and 213,995, respectively (32).According to the report from the Ethiopian Public Health Institute of 2023, among a total of 571, 119 adult PLHIV in Ethiopia, 107,496 were from Addis Ababa, while 6,300 were from Adama (33).In Ethiopia, 98% of healthcare settings, including all referral hospitals, provide comprehensive HIV care services (34).

Estimation of sample size and recruitment of study participants
The sample size was estimated using the formula ], where Z is the standardized score at a 97% confidence interval (CI), e = margin of error, P = is the estimated proportion of the population with UTIs in the target community, and N = total estimated sample size.Thus, considering a 10.3% proportion (4), 97% CI, 0.03 margin of error, and 7.5% non-response rate, the total estimated sample size was 520 for the study population in Addis Ababa, and considering an 11.3% proportion (26), 95% CI, 5% margin of error, with the non-response rate (5%), and the total sample size calculated was 170 for Adama.The total sample size for the two cities was 688.The studied populations were PLHIV attending ART clinics of selected referral hospitals providing comprehensive HIV care; The exclusion criteria include those who consumed two glasses of fluid an hour before attending the clinic, as fluid intake dilutes and reduces bacteria concentration in the urine (35), (b) received antibiotic treatment within a week before attending the clinic, which might affect UTI test result (36), (c) those with confirmed sexually transmitted diseases (STDs) who exhibit symptoms overlapping with UTIs (37), (d) pregnant or lactating mothers who might have immune suppression and an excess risk of having UTI (38, 39), menstruating women, could be due to the considerable lowering level of estrogen during the menstrual period (40), and the blood may contaminate urine samples which can affect UTI testing results, (e) individuals <18 years old, as this study focused on adult patients.Then, the estimated sample size was proportionally allocated across the selected seven ART clinics based on the total number of PLHIV attending the clinics during the study period, and a simple random sampling method was used to select study participants using the list of PLHIV within each clinic as a sampling frame.

Data collection methods
Prospective background characteristics were collected through face-to-face interviews, while clinical characteristics were determined by reviewing recent information sheets of the patients' charts.The meta-data and urine samples were collected using an expert-validated, pretested structured questionnaire adapted from related literature (26,28), and checked for content validity index (CVI) rated at 0.82; indicating a high level of relevance, and appropriateness of the questionnaire in measuring the intended constructs.The data collectors were well-qualified nurses who received 2 days of training on research methodology, and how to collect the meta-data, and urine samples credibly.A total of 10 mL of midstream clean catch urine samples were collected in sterile urine cups with cover through precautions to prevent contamination and transported to the microbiology laboratory of Aklilu Lemma Institute of Pathobiology within 4 h of collection, maintaining its cold chain.
Isolates resistant to at least one in three or more antibiotic categories were considered multi-drug resistant (MDR) and being resistant to at least one to two or fewer antibiotic categories were considered extensive drug resistant (XDR), while those resistant to all antibiotic categories tested were considered pan-drug-resistant (PDR) (47,48).The multiple antibiotic resistance index (MARI) was calculated as MARI = a/b, which is the ratio of the number of antibiotics to which an isolate is resistant (a) to the total number of antibiotics to which an isolate is tested (b) (49).Comprehensive explanations were given to the study participants about the objective of the study in a private room prepared for interview, examination, and chart review before obtaining written consent.The study participants were assured not to participate and/or discontinue the interview at any point in time.The data collected was stored in a locker, which can only be accessed by the principal investigator.We assured and implemented that those found culture positive for UTIs were treated for the infection under the follow-up institution and assigned health care provider.

Data management and analysis
The collected data were entered into Epi-data version 3.1 and exported to SPSS version 23 for analysis.Figures and tables were used to summarize the data.To measure the association of variables with the occurrence of UTIs, we used univariable and multivariable logistic regression analyses.Crude odds ratios (CORs) were obtained from the univariable regression analysis, while adjusted odds ratios (AORs) were derived from the multivariable regression analysis.The results from the final models were presented as AOR with a 95% CI, with association determined at a threshold of a p-value of <0.05.
A total of 218 participants (31.7%) had a history of receiving antituberculosis drugs, of which 84 (38.5%) were cured, while the remaining were still on treatment.Moreover, substantial (n = 51; 7.4%) of the study participants were receiving zidovudine-containing antiretroviral drug regimens (Table 2).In total, 59 (8.6%) of the study participants had a body mass index of less than 18.5wt/ht 2 , implying undernutrition (Table 2).Furthermore, the majority of the study participants 645(93.7%)were found under T1 World Health Organization clinical staging of HIV/AIDS, which implies that the viral load of these PLHIV was significantly reduced after starting the ART drugs (Table 2).

Prevalence of UTIs and its associated factors
Out of a total of 688 study participants (n = 144; 20.9%) were found to have acquired UTIs confirmed by bacterial, culture.Of those who acquired UTIs, 36 participants (69.2%) were symptomatic, with the most prevalent symptom being dysuria (n = 14; 63.6%) (Table 1).
The presentation of clinical characteristics in relation to the prevalence of UTIs, among those who acquired UTIs (n = 144; 20.9%), a significant population (n = 9; 75.0%) had HIV RNA of ≥200 copies/ ml, and the majority (n = 136; 21.1%) were in stage T1 of the World Health Organization clinical staging of HIV/AIDS.The higher (n = 12; 23.5%) proportions were from those who were receiving zidovudinecontaining ART drug regimen of HIV treatment compared to those who were taking non-zidovudine-containing ART drug (Table 2).

Prevalence of bacterial isolates causing UTIs
A total of 151 bacterial isolates were isolated from 144 study participants who had UTIs, which implies seven study participants who acquired UTIs were infected by more than one bacterial causal agent.The most frequently occurring bacterial isolates together were E. coli and Bacillus cereus.E. coli (n = 65; 45.1%) was the most common bacterial isolate identified followed by E. faecalis (n = 16;  1).Cross-tabulating the bacterial isolates rarely causes UTIs to selected study variables, the single bacterial isolates: Salmonella species, Fusobacterium nucleatum, Klebsiella aerogenes, Pseudomonas fulva, Alcaligenes faecalis, Cronobacter sakazakii, Enterobacter asburiae, Enterobacter amnigenus, Staphylococcus pasteuri, and E. hirae, were identified from study participants with older ages (35 years or older), living in urban areas, having history of chronic illness such as diabetes mellitus, hypertension, renal caliculi, and previous history of UTIs, no/less frequently practicing vaginal or perineal care, and currently exhibiting symptoms of UTIs.Conversely, Alcaligenes feacalis, Fusobacterium nucleatum, Enterobacter asburiae, Enterobacter amnigenus, and E. hirae were identified among asymptomatic study participants for UTIs.
Additionally, of the bacterial isolates that were found causing UTIs in this study, approximately one-third (n = 104; 68.9%) were Gram-negative, with the remainder being Gram-positive (Figure 1).

Antibiotic resistance pattern and multiple antibiotic resistance index
The study revealed, that among Gram-negative bacterial isolates, all K. pneumoniae, Enterobacter cloacae, and Enterobacter bugandensis isolates were resistant to amoxicillin + clavulanic acid.All isolates of Enterobacter bugandensis were resistant to ciprofloxacin, azithromycin, and gentamicin.All P. mirabilis isolates were resistant to amoxicillin (Table 3; Supplementary Table 1).
All S. aureus isolates were resistant to cefoxitin, amoxicillin, amoxicillin + clavulanic acid, and clindamycin.Additionally, over half of these isolates were resistant to ciprofloxacin, ceftriaxone, kanamycin, and cephalothin.These resistance patterns indicate that all S. aureus isolates in the study were methicillin-resistant, highlighting a significant challenge in treating infections caused by these bacteria (Table 3; Supplementary Table 1).
All bacterial isolates in the study were resistant to at least one antibacterial agent, with the highest multiple antimicrobial resistance index (MARI) observed among E. faecalis, S. xylosus, and E. coli.This indicates significant variability in ABR among the isolates (Supplementary Table 1).
A total of 138 isolates (91.4%) were resistant to at least one antibiotic in three or more categories, indicating MDR.Of these, 43 isolates (31.2%) were resistant to all but two or three of the antibiotic categories tested, classifying them as XDR.The high variability in the MARI among the isolates that developed MDR and XDR highlights a concerning trend in the increasing complexity and capriciousness of ABR (Supplementary Table 1).
Higher HIV RNA copies and the manifestation of UTI symptoms were found to be positively associated with the occurrence of UTIs.The most frequently identified bacterial uropathogen was E. coli, followed by E. faecalis and K. pneumoniae.Additionally, several rare UTI-causing bacterial isolates were identified, which exhibited high levels of ABR.
The link between higher levels of HIV RNA copies and the occurrence of UTIs highlights the intricate relationship between viral load and susceptibility to opportunistic infections, including UTIs.Elevated viral load often correlates with higher viral replication and a more active HIV infection, leading to the progressive depletion of CD4 cells.This results in a weakened immune system, increasing the risk of opportunistic infections.Various factors can influence this susceptibility, such as co-infections with other viruses, genetic factors, geographic location, lifestyle choices, nutrition, stress, and pregnancy, all of which contribute to a greater risk of acquiring infections such as UTIs (2).
Furthermore, the link between clinical symptoms of UTIs and the occurrence of UTIs may be attributed to the irritation of the bladder trigone or urethra.This irritation causes the bladder to contract, resulting in more frequent and painful urination (52).
The distribution of bacterial isolates causing UTIs revealed in the current study shows approximately three-fourths (n = 104; 68.9%) were Gram-negative organisms.This distribution aligns with study findings from Ethiopia (28), South Africa (9), and Uganda (53).However, studies from other regions in Nigeria (19) and India (54) reported Gram-positive bacteria as the most prevalent UTI-causing bacteria among PLHIV.This variation could be due to differences in geographic location, characteristics of the study population, and the laboratory methodologies used for bacterial isolation in the different regions.The frequent isolation of E. coli could be due to certain strains, known as uropathogenic E. coli (UPEC), possessing specialized virulence factors.These include adhesins (fim and pap operons), toxins (hemolysin and cytotoxic-necrotizing factors), and siderophores (fyuA gene), which facilitate their ability to cause UTIs.These virulence factors enable UPEC to adhere to the urinary tract epithelium, evade the host immune response, and acquire essential nutrients, thereby increasing their pathogenic potential (2).This finding aligns with studies conducted globally (55), as well as in Ethiopia (28), Nigeria (56), Uganda (53), and Croatia (57).However, contrary to our study, findings from Nigeria (19), Ghana (24), and India (54) revealed that the most frequently isolated bacterial species causing UTIs was S. aureus.
The identification of E. faecalis as the second most frequent bacterial species in this study, with a higher prevalence of ABR, could be due to its significant impact on vulnerable populations, such as those living with HIV (58).This pathogen's resilience and adaptability make it particularly problematic in immunocompromised individuals, contributing to its prominence in such settings (58).This finding is consistent with studies from Tanzania (27) and Croatia (57), raising concerns regarding public health implications, especially in areas with limited access to effective antibiotics.The prevalence of E. faecalis and its resistance to antibiotics highlights the need for improved infection control measures and the development of more effective treatment strategies in such regions.
The less frequently isolated bacterial species causing UTIs reported in the current study raise concerns about the emergence of new pathogens contributing to UTI occurrence.This issue is significant for global public health, as it highlights the need for ongoing vigilance and research.The scientific community needs to investigate these emerging bacteria, understand their pathogenic mechanisms, and develop effective treatment and preventive strategies to address the evolving landscape of UTI pathogens.
The higher prevalence of bacterial uropathogens, including rarely identified pathogens causing UTIs, as revealed by studies in the region, highlights growing concerns about increasing ABR globally.This escalating threat of antimicrobial resistance (AMR) poses a substantial challenge to public health, particularly among PLHIV in sub-Saharan African countries where resources are limited (59).Addressing this issue requires a multi-faceted approach: enhanced surveillance, antibiotic stewardship, infection control measures, research and development, public awareness and education, strengthening healthcare systems, and global collaboration.
The finding that all bacterial isolates were resistant to at least one antibacterial agent, along with the high levels of MDR, underscores the gravity of the problem.This situation is concerning as it implies  Frontiers in Public Health 09 frontiersin.orglimited treatment options, an increased risk of complications, and significant public health implications.The higher prevalence of ABR observed in the current study among E. coli isolates to various antibiotics: ciprofloxacin (61.5%), azithromycin (69.2%), and nalidixic acid (64.6%).This finding aligns with studies from Nigeria (50) and Ethiopia (4), indicating a broadening issue of AMR.
However, the variation in resistance patterns, such as the lower resistance rate reported in Harar, Ethiopia (20.5%) (22), highlights the multifactorial nature of AMR.Differences in resistance patterns across regions can be attributed to factors such as local antibiotic prescribing practices, infection control measures, healthcare infrastructure, and the prevalence of resistance genes.This highlights the need for region-specific strategies to monitor and address resistance trends effectively.
The observed high prevalence of ABR among P. mirabilis to various antibiotics, such as amoxicillin, amoxicillin+clavulanic acid, and gentamicin, aligns with study findings from Nigeria (50) and Ethiopia (22).This widespread resistance poses significant challenges for treatment.However, the contrasting results from India (60) highlight the variability of antibiotic resistance patterns across different regions.This variability highlights the importance of local surveillance and tailored treatment strategies to effectively manage and address antibiotic resistance in different settings.
The World Health Organization has identified MRSA as a significant pathogen that poses serious health risks (61).MRSA alone was responsible for over 100,000 deaths in 2019 (62).The current study found that all S. aureus isolates were resistant to cefoxitin, indicating a 100% prevalence of MRSA.This rate is notably higher than the 27.9% prevalence reported in Ireland (63).
The high prevalence of MRSA in this study may be attributed to factors such as the inappropriate use of antibiotics, which can drive the development and spread of resistant strains.This highlights the urgent need for improved antibiotic stewardship and infection control measures.
This study highlights a crucial aspect that could further enhance its comprehensiveness, investigating other potential causative agents of UTIs beyond bacterial isolates.By including viruses and fungi, researchers could gain a more complete understanding of the prevalence, etiology, and management of UTIs among PLHIV.Such an approach would provide a broader perspective on the range of pathogens involved, potentially leading to more effective diagnostic and treatment strategies.

Conclusion
The current study revealed a high prevalence of UTIs and elevated levels of ABR as compared to similar studies in other regions.Independent predictors for UTI occurrence identified in the study include higher HIV RNA copies and the manifestation of UTI symptoms during the study period.The predominant bacterial isolates in this population were E. coli, K. pneumoniae, and E. faecalis.This finding highlights the need for targeted surveillance, raising public awareness, integrating UTI screening programs for PLWH, establishing measures for responsible antibiotic use and antibiotic stewardship programs, and tailoring treatment strategies to address the unique challenges posed by these pathogens and resistance patterns in the study population.The study also found several rare UTI-causing bacterial species which need to be investigated.
The Institutional Research Ethics Review Board of the College of Health Sciences (IRERB Minutes Ref No.: AAUMF 03-008) and Aklilu Lemma Institute of Pathobiology (IRERB Minutes Ref No.: ALIPB IREC/86/14/22) Addis Ababa University reviewed the protocol and provided ethical clearance letter.

TABLE 1
Background characteristics of study participants and association with UTIs, N = 688.

TABLE 2
Clinical characteristics and association with the occurrence of UTIs, N = 688.Salmonella species, demonstrated resistance to amoxicillin with clavulanic acid as well as other broad-spectrum antibiotics, including ciprofloxacin, ceftriaxone, azithromycin, gentamicin, and nalidixic acid (Table3; Supplementary Table1).

TABLE 3
Antimicrobial susceptibility profile of bacterial strains isolated from urine culture-positive study participants, N = 151.